姜黄素改善高糖无血清环境下骨髓间充质干细胞的损伤及旁分泌作用

Curcumin Improves the Damage and Paracrine Effects of BMSCs in a High Glucose and Serum Free Environment

目的:探讨姜黄素对高糖无血清环境下骨髓间充质干细胞(BMSCs)损伤的保护作用及旁分泌的影响。方法:采用贴壁筛选培养法分离纯化SD大鼠骨髓间充质干细胞,对其进行成骨、成脂分化及流式鉴定;取第3代细胞用于实验。采用CCK8检测姜黄素细胞毒性;显微镜下观察细胞形态变化;采用流式细胞术分析细胞凋亡;倒置荧光显微镜检测细胞内活性氧(ROS)水平;ELISA法检测血管内皮生长因子(VEGF)、血小板源性生长因子(PDGF)含量水平。结果:与空白对照组相比,姜黄素1μmol/L~10μmol/L组对正常BMSCs不具有毒性作用(P>0.05);与空白对照组相比,模型对照组BMSCs形态改变,凋亡率显著增加(P<0.01),细胞内ROS含量明显增加(P<0.05),VEGF、PDGF含量明显降低(P<0.05或P<0.01);与模型对照组相比,姜黄素浓度在1μmol/L~10μmol/L可改善BMSCs受损形态,姜黄素10μmol/L组细胞凋亡率、细胞内ROS的含量降低(P<0.05或P<0.01),姜黄素5、10μmol/L组VEGF、PDGF含量明显增加(P<0.05或P<0.01)。结论:姜黄素对高糖无血清环境下的BMSCs损伤具有良好保护作用,可能与降低细胞内氧化应激,减轻细胞凋亡相关,并最终改善细胞旁分泌功能。

Objective: To investigate the effects of curcumin on the damage and paracrine of bone marrow mesenchymal stem cells(BMSCs) in a high glucose and serum-free environment. Methods: Adherent screening culture method was used to isolate and purify SD rat bone marrow mesenchymal stem cells, and then to perform osteogenic, adipogenic differentiation and flow cytometric identification. The third generation cells were used for the experiment. CCK8 was used to detect the curcumin cytotoxicity, the cell morphology changes were observed under a microscope, the flow cytometry was used to analyze the cell apoptosis. Inverted fluorescence microscope was used to detect the intracellular reactive oxygen species(ROS) level. ELISA was used to detect the expression levels of vascular endothelial growth factor(VEGF) and platelet-derived growth factor(PDGF). Results: Compared with the control group, Curcumin had no toxic effect on normal BMSCs at the doses of 1 μmol/L~10 μmol/L(P>0.05), the morphology of BMSCs was changed in the model group, the cell apoptosis was increased(P<0.01),the content of intracellular ROS was significantly increased(P<0.05), the contents of VEGF and PDGF were significantly decreased(P<0.05 or P<0.01). Compared with the model group, the concentrations of curcumin at 1 μmol/L~10 μmol/L improved the damaged morphology of BMSCs. 10 μmol/L curcumin reduced the content of intracellular ROS and the rate of cell apoptosis(P<0.05 orP<0.01),5 and 10 μmol/L Curcumin increased the contents of VEGF and PDGF(P<0.05 or P<0.01).Conclusion: Curcumin has a good protective effect on BMSCs damage in a high glucose and serum-free environment, which may be related to reducing the intracellular oxidative stress, inhibiting the cell apoptosis, and finally improving paracrine function.